Negative strand RNA viruses (NRVs), such as vesicular stomatitis virus (VSV), are unique because their nucleocapsid, not the naked RNA, is the active template for transcription and replication. During the complete infection cycle, the genomic RNA is completely sequestered by the nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRp, a complex between L and P) must gain access to the bases of the RNA in order to initiate transcription or replication. Our previous structural studies showed how the RNA is encapsidated by the nucleocapsid, as well as the structure and accessibility of the RNA within the encapsidation cavity. Since the nucleocapsid is the template for viral RNA synthesis and given the intimate association between N and the RNA, questions arise as to what role the N protein may play in transcription and replication. The new studies proposed here are focused on: Aim 1, the structural requirement of the functional template in initiation of viral transcription and replication. We have developed methods to solve the structure of specific RNA sequences encapsidated within nucleocapsid-like particles (NLPs). In this aim, we plan to solve a novel series of structures aimed toward addressing the question about how the N protein helps RdRp to recognize specific RNA sequences sequestered in the nucleocapsid. This will be complemented with a look at structural changes in the N protein that affect transcription/replication. This will be accomplished by studying a series of mutant N proteins with phenotypes that affect these enzymatic processes. These mutants suggest that the N protein itself plays a role in regulation of transcription/replication. In Aim 2, we will examine polynucleotide synthesis from the role of the L and P proteins, rather than the functional template. We have engineered a series of L protein fragments that are soluble, two of which have been crystallized. We will determine structures for several domains of L. These will be integrated with EM studies of the L, P and N proteins aimed at reconstructing the larger tripartite replicase complex. Collectively, the studies proposed here will address both replication and transcription from two perspectives, the template as well as the machinery involved in this critical enzymatic process.